Amicon ultra 100 k cutoff spin concentrators

Purification of needle complexes from MIB3118 (InvC_K165E) was performed as described previously 46 (link). This InvC mutant disrupts the ATPase activity in order to make the system secretion incompetent without disrupting other components and it mimics the approach taken to produce the 6.3 ÅS. Typhimurium basal body cryo-EM map 48 (link). After collection from the CsCl-gradient, needle complexes were subjected to size exclusion chromatography (Superdex 200 10/300 GL, GE Healthcare) and collected from the void volume. Needle complexes were concentrated to 1 mg/ml using Amicon Ultra 100 k cutoff spin concentrators (Merck Millipore) and subsequently subjected to chemical cross-linking. Disuccinimidyl suberate (DSS; 50 mM; Thermo Fisher Scientific) in DMSO was added to a final concentration of 1 mM to the complex solution of 20 µg/ml. The reaction mixture was incubated with agitation at 4 °C for 3 h. The reaction was quenched by addition of 1 M Tris-HCl, pH 7.5, to a final concentration of 50 mM for 15 min at room temperature. The crosslinked needle complexes were analyzed by SDS PAGE and Coomassie staining. For mass spectrometry, the crosslinked needle complex was run 1 cm into a 4-20% SDS PAGE gel, stained by Coomassie, and all stained material in the lane was cut out for further analysis.